丙酮酸乙酯抑制LPS诱导RAW264.7巨噬细胞HMGB1的表达

Ethyl pyruvate inhibited HMGB1 expression induced by LPS in macrophages

目的:探讨丙酮酸乙酯(EP)抑制脂多糖(LPS)诱导巨噬细胞高迁移族率蛋白B1(HMGB1)的表达及分子机制。方法:培养小鼠巨噬细胞株RAW264.7,分为LPS(100 ng/mL)组及LPS(100 ng/mL)+EP(5 mmol/L)组,刺激后0、6、12、18、24、30、36 h提取总RNA及胞质胞核蛋白,用RT-PCR法测细胞培养液中HMGB1 mRNA表达;用Western blot测胞质和胞核HMGB1蛋白含量;用ELISA法测培养上清中HMGB1、TNF-α和IL-6的含量;用免疫细胞化学共聚焦显微镜观察细胞内HMGB1的转位分布。结果:LPS+EP组HMGB1 mRNA基因表达于24、36、48 h比LPS组明显减少;Western blot结果示,LPS+EP组HMGB1蛋白含量胞质明显低于LPS组,而胞核明显高于LPS组;ELISA结果示,LPS+EP组细胞培养上清中HMGB1、TNF-α和IL-6的含量明显低于LPS组;免疫荧光、激光共聚焦显微镜结果示,LPS+EP组细胞质内绿色荧光染色明显弱于LPS组,细胞核内绿色荧光染色明显强于LPS组。结论:EP能有效抑制LPS诱导的小鼠腹腔巨噬细胞HMGB1的表达和释放。

AIM： To elucidate the mechanism of ethyl pyruvate （EP） inhabit high mobility group protein B1 （HMGB1） expression and releasing in macrophage induced by lipopolysaccharide（LPS）. METHODS： The murine macrophage-like cell line RAW264.7 cultured in vitro divided into LPS group and LPS ＋ EP group. The expression of HMGB1 mRNA in cultured cell was determined by RT-PCR, The cytoplasmic and nuclear HMGB1 levels were detected by Western blot. The contents of HMGB1 and TNF-α and IL-6 protein in cultured cells supernatant were detected by ELISA. Immunocytochemistry and confocal laser-scanning microscopy were used to confirm the relocation and distribution of intracellular HMGB1 protein in RAW264.7 cells. RESULTS： HMGBI mRNA expression in the LPS ＋ EP group was significantly lower than in LPS alone, at 24, 36 and 48 hours. In the LPS ＋ EP stimulation group, the cytoplasm stained weakly while the nuclear stain was stronger than that of the LPS group at the same time points. Both TNF-α and IL-6 levels in LPS ＋ EP group were significantly lower than those in the LPS group at the same time points. EP also effectively prevented the release of HMGB1 protein. CONCLUSION： EP inhibits HMGB1 expression and release from LPS-stimulated macrophages.